Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with a number of cancers including Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin lymphoma, and post-transplant lymphoproliferative disease. EBV infects B cells and 95% of adults are infected. We have developed nanoparticles which contain EBV glycoproteins and have shown that they are immunogenic in mice and nonhuman primates. These EBV nanoparticles are being produced for clinical trials at the NIH Clinical Center. Human cytomegalovirus (HCMV) infects over half of the human population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. A human cytomegalovirus (HCMV) vaccine to prevent infection and/or reduce disease associated with congenital infection or visceral disease in transplant recipients is a high priority, but has remained elusive. We have produced a replication-defective rhesus CMV (RhCMV) vaccine and have compared it with a RhCMV subunit vaccine in rhesus monkeys for its ability to produce virus-specific antibodies, T cells, and to protect against challenge with wild-type rhesus CMV. The replication-defective RhCMV vaccine is deleted for glycoprotein L (gL) and the MHC class I immune evasion genes Rh178 and Rh182-189. We restored its epithelial cell tropism by inserting the Rh128-131A genes. The resulting virus, RhCMVRgL/178/182-189, was used to vaccinate rhesus monkeys intramuscularly and was compared with vaccination of animals with soluble RhCMV glycoprotein B (gB) in alum/monophosphoryl lipid A or with PBS as a control. At 4 weeks after the second vaccination, an increased frequency of RhCMV-specific CD8 T cells was detected in animals vaccinated with the RhCMVRgL/178/182-189 vaccine compared to animals vaccinated with soluble gB. In contrast, monkeys vaccinated with soluble gB had 20-fold higher gB antibody titers than animals vaccinated with RhCMVRgL/178/182-189. Titers of neutralizing antibody to RhCMV infection of fibroblasts were higher in animals vaccinated with gB compared with RhCMVRgL/178/182-189. Following vaccination, monkeys were challenged subcutaneously with RhCMV UCD59, a low passage virus propagated in monkey kidney epithelial cells. All animals became infected after challenge; however, the frequency of RhCMV detection in the blood was reduced in monkeys vaccinated with soluble gB compared with those vaccinated with RhCMVRgL/178/182-189. The frequency of challenge virus shedding in the urine and saliva and the RhCMV copy number shed at these sites was not different in animals vaccinated with RhCMVRgL/178/182-189 or soluble gB compared with those that received PBS before challenge. Although the RhCMVRgL/178/182-189 vaccine was superior in inducing cellular immunity to RhCMV, it induced lower titers of neutralizing antibody and antibody to gB than the soluble gB vaccine; after challenge, animals vaccinated with soluble gB had a lower frequency of virus detection in the blood than those vaccinated with RhCMVRgL/178/182-189.